To induce antigen-specific T cell activation and clonal expansion, two signals provided by antigen-presenting cells (APCs) must be delivered to the surface of resting T lymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp. Med. 165:302-319; Mueller, D. L., et al. (1990) J. Immunol. 144:3701-3709; Williams, I. R. and Unanue, E. R. (1990) J. Immunol. 145:85-93). The first signal, which confers specificity to the immune response, is mediated via the T cell receptor (TCR) following recognition of foreign antigenic peptide presented in the context of the major histocompatibility complex (MHC). The second signal, termed costimulation, induces T cells to proliferate and become functional (Schwartz, R. H. (1990) Science 248:1349-1356). Costimulation is neither antigen-specific, nor MHC restricted and is thought to be provided by one or more distinct cell surface molecules expressed by APCs (Jenkins, M. K., et al. (1988) J. Immunol. 140:3324-3330; Linsley, P. S., et al. (1991) J. Exp. Med. 173:721-730; Gimmi, C. D., et al., (1991) Proc. Natl. Acad. Sci. USA. 88:6575-6579; Young, J. W., et al. (1992) J. Clin. Invest. 90:229-237; Koulova, L., et al. (1991) J. Exp. Med. 173:759-762; Reiser, H., et al. (1992) Proc. Natl. Acad. Sci. USA. 89:271-275; van-Seventer, G. A., et al. (1990) J. Immunol. 144:4579-4586; LaSalle, J. M., et al., (1991) J. Immunol. 147:774-80; Dustin, M. I., et al., (1989) J. Exp. Med. 169:503; Armitage, R. J., et al. (1992) Nature 357:80-82; Liu, Y., et al. (1992) J. Exp. Med. 175:437-445).
Considerable evidence suggests that the B7-1 protein (CD80; originally termed B7), expressed on APCs, is one such critical costimulatory molecule (Linsley, P. S., et al., (1991) J. Exp. Med. 173:721-730; Gimmi, C. D., et al., (1991) Proc. Natl. Acad Sci. USA. 88:6575-6579; Koulova, L., et al., (1991) J. Exp. Med. 173:759-762; Reiser, H., et al. (1992) Proc. Natl. Acad Sci. USA. 89:271-275; Linsley, P. S. et al. (1990) Proc. Natl. Acad. Sci. USA. 87: 5031-5035; Freeman, G. J. et al. (1991) J. Exp. Med. 174:625-631.). Recent evidence suggests the presence of additional costimulatory molecules on the surface of activated B lymphocytes (Boussiotis V. A., et al. (1993) Proc. Natl. Acad Sci. USA, 90:11059-11063; Freeman G. J., et al. (1993) Science 262:907-909; Freeman G. J., et al. (1993) Science 262:909-911; and Hathcock K. S., et al. (1993) Science 262:905-907). The human B lymphocyte antigen B7-2 (CD86) has been cloned and is expressed by human B cells at about 24 hours following stimulation with either anti-immunoglobulin or anti-MHC class II monoclonal antibody (Freeman G. J., et al. (1993) Science 262:909-911). At about 48 to 72 hours post activation, human B cells express both B7-1 and a third CTLA4 counter-receptor which is identified by a monoclonal antibody BB-1, which also binds B7-1 (Yokochi, T., et al. (1982) J. Immunol. 128:823-827). The BB-1 antigen is also expressed on B7-l negative activated B cells and can costimulate T cell proliferation without detectable IL-2 production, indicating that the B7-1 and BB-1 molecules are distinct (Boussiotis V. A., et al. (1993) Proc. Natl. Acad Sci. USA 90:11059-11063). The presence of these costimulatory molecules on the surface of activated B lymphocytes indicates that T cell costimulation is regulated, in part, by the temporal expression of these molecules following B cell activation.
B7-1 is a counter-receptor for two ligands expressed on T lymphocytes. The first ligand, termed CD28, is constitutively expressed on resting T cells and increases after activation. After signaling through the T cell receptor, ligation of CD28 induces T cells to proliferate and secrete IL-2 (Linsley, P.S., et al. (1991) J. Exp. Med. 173: 721-730; Gimmi, C. D., et al. (1991) Proc. Natl. Acad. Sci. USA. 88:6575-6579; Thompson, C. B., et al. (1989) Proc. Natl. Acad. Sci. USA. 86:1333-1337; June, C. H., et al. (1990) Immunol. Today. 11:211-6; Harding, F. A., et al. (1992) Nature. 356:607-609.). The second ligand, termed CTLA4, is homologous to CD28, but is not expressed on resting T cells and appears following T cell activation (Brunet, J. F., et al., (1987) Nature 328:267-270). Like B7-1, B7-2 is a counter-receptor for both CD28 and CTLA4 (Freeman G. J., et al. (1993) Science 262:909-911). CTLA4 was first identified as a mouse cDNA clone, in a library of cDNA from a cytotoxic T cell clone subtracted with RNA from a B cell lymphoma (Brunet, J. F., et al. (1987) supra). The mouse CTLA4 cDNA was then used as a probe to identify the human and mouse CTLA4 genes (Harper, K., et al. (1991) J. Immunol. 147:1037-1044; and Dariavich, et al. (1988) Eur. J. Immunol. 18(12):1901-1905, sequence modified by Linsley, P. S., et al. (1991) J. Exp. Med. 174:561-569). A probe from the V domain of the human gene was used to detect the human cDNA which allowed the identification of the CTLA4 leader sequence (Harper, K., et al. (1991) supra).
Soluble derivatives of cell surface glycoproteins in the immunoglobulin gene superfamily have been made consisting of an extracellular domain of the cell surface glycoprotein fused to an immunoglobulin constant (Fc) region (see e.g., Capon, D. J. et al. (1989) Nature 337:525-531 and Capon U.S. Pat. Nos. 5,116,964 and 5,428,130 [CD4-IgG1 constructs]; Linsley, P. S. et al. (1991) J. Exp. Med. 173:721-730 [a CD28-IgG1 construct and a B7-1-IgG1 construct]; and Linsley, P. S. et al. (1991) J. Exp. Med. 174:561-569 and U.S. Pat. No. 5,434,131 [a CTLA4-IgG1). Such fusion proteins have proven useful for studying receptor-ligand interactions. For example, a CTLA4-IgG immunoglobulin fusion protein was used to study interactions between CTLA4 and its natural ligands (Linsley, P. S., et al., (1991) J. Exp. Med. 174:561-569; International Application WO93/00431; and Freeman G. J., et al. (1993) Science 262:909-911).
The importance of the B7: CD28/CTLA4 costimulatory pathway has been demonstrated in vitro and in several in vivo model systems. Blockade of this costimulatory pathway results in the development of antigen specific tolerance in murine and human systems (Harding, F. A., et al. (1992) Nature 356:607-609; Lenschow, D. J., et al. (1992) Science 257:789-792; Turka, L. A., et al. (1992) Proc. Natl. Acad. Sci. USA 89:11102-11105; Gimmi, C. D., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6586-6590; Boussiotis, V., et al. (1993) J. Exp. Med. 178:1753-1763). Conversely, transfection of a B7-1 gene into B7-1 negative murine tumor cells to thereby express B7-1 protein on the tumor cell surface induces T-cell mediated specific immunity accompanied by tumor rejection and long lasting protection to tumor challenge (Chen, L., et al. (1992) Cell 71: 1093-1102; Townsend, S. E. and Allison, J. P. (1993) Science 259:368-370; Baskar, S., et al. (1993) Proc. Nati. Acad. Sci. USA 90:5687-5690.). Therefore, approaches which manipulate the B7: CD28/CTLA4 interaction to thereby stimulate or suppress immune responses would be beneficial therapeutically.
This invention pertains to CTLA4-immunoglobulin fusion proteins having modified immunoglobulin constant (IgC) region-mediated effector functions and to nucleic acids encoding the proteins. In one embodiment, the fusion proteins of the present invention have been constructed by fusing a peptide having a CTLA4 activity and a second peptide comprising an immunoglobulin constant region to create a CTLA4Ig fusion protein. In another embodiment, the variable regions of immunoglobulin heavy and light chains have been replaced by the B7-binding extracellular domain of CTLA4 to create CTLA4-Ab fusion proteins. As used herein, the term xe2x80x9cCTLA4-immunoglobulin fusion proteinxe2x80x9d refers to both the CTLA4Ig and CTLA4-Ab forms. In a preferred embodiment, the fusion proteins of the invention have been modified to reduce their ability to activate complement and/or bind to Fc receptors. In one embodiment, an IgC region of an isotype other than Cxcex31 is used in the fusion protein and the modified effector function(s) can be assessed relative to a Cxcex31-containing molecule (e.g., an IgG1 fusion protein). In another embodiment, a mutated IgC region (of any isotype) is used in the fusion protein and the modified effector function can be assessed relative to an antibody or Ig fusion protein containing the non-mutated form of the IgC region.
The CTLA4-immunoglobulin fusion proteins of the invention are useful for inhibiting the interaction of CTLA4 ligands (e.g., B7 family members such as B7-1 and B7-2) with receptors on T cells (e.g., CD28 and/or CTLA4) to thereby inhibit delivery of a costimulatory signal in the T cells and thus downmodulate an immune response. Use of the CTLA4-immunoglobulin fusion proteins of the invention is applicable in a variety of situations, such as to inhibit transplant rejection or autoimmune reactions in a subject. In these situations, immunoglobulin constant region-mediated biological effector mechanisms, such as complement-mediated cell lysis, Fc receptor-mediated phagocytosis or antibody-dependent cellular cytotoxicity, may induce detrimental side effects in the subject and are therefore undesirable. The CTLA4-immunoglobulin fusion proteins of the invention exhibit reduced IgC region-mediated effector functions compared to a CTLA4-immunoglobulin fusion protein in which the IgG1 constant region is used and, thus are likely to have improved immunoinhibitory properties. These compositions can also be used for immunomodulation, to produce anti-CTLA4 antibodies, to purify CTLA4 ligands and in screening assays. The CTLA4-Ab fusion proteins are particularly useful when bivalent preparations are preferred, i.e. when crosslinking is desired.
One aspect of the invention pertains to isolated nucleic acid molecules encoding modified CTLA4-immunoglobulin fusion proteins. The nucleic acids of the invention comprise a nucleotide sequence encoding a first peptide having a CTLA4 activity and a nucleotide sequence encoding a second peptide comprising an immunoglobulin constant region which is modified to reduce at least one constant region-mediated biological effector function. A peptide having a CTLA4 activity is defined herein as a peptide having at least one biological activity of the CTLA4 protein, e.g., the ability to bind to the natural ligand(s) of the CTLA4 antigen on immune cells, such as B7-1 and/or B7-2 on B cells, or other known or as yet undefined ligands on immune cells, and inhibit (e.g., block) or interfere with immune cell mediated responses. In one embodiment, the peptide having a CTLA4 activity binds B7-1 and/or B-2 and comprises an extracellular domain of the CTLA4 protein. Preferably, the extracellular domain includes amino acid residues 20-144 of the human CTLA4 protein (amino acid positions 20-144 of SEQ ID NO: 24, 26 and 28).
The present invention also contemplates forms of the extracellular domain of CTLA4 which are expressed without Ig constant regions and are expressed in E. coli. These soluble forms of the CTLA4 extracellular domain, although not glycosylated, are fully functional and have similar uses as the CTLA4 immunoglobulin fusion proteins of the invention.
The nucleic acids of the invention further comprise a nucleotide sequence encoding a second peptide comprising an immunoglobulin constant region which is modified to reduce at least one Ig constant region-mediated biological effector function. Preferably, the immunoglobulin constant region comprises a hinge region, a CH2 domain and a CH3 domain derived from Cxcex31, Cxcex32, Cxcex33 or Cxcex34. In one embodiment, the constant region segment is altered (e.g., mutated at specific amino acid residues by substitution, deletion or addition of amino acid residues) to reduce at least one IgC region-mediated effector function. In another embodiment, a constant region other than Cxcex31 that exhibits reduced IgC region-mediated effector functions relative to Cxcex31 is used in the fusion protein. In a preferred embodiment, the CH2 domain is modified to reduce a biological effector function, such as complement activation, Fc receptor interaction, or both complement activation and Fc receptor interaction. For example, to reduce Fc receptor interaction, at least one amino acid residue selected from a hinge link region of the CH2 domain (e.g., amino acid residues at positions 234-237 of an intact heavy chain protein) is modified by substitution, addition or deletion of amino acids. In another embodiment, to reduce complement activation ability, a constant region which lacks the ability to activate complement, such as Cxcex34 or Cxcex32 is used in the fusion protein (instead of a Cxcex31 constant region which is capable of activating complement). In another embodiment the variable region of the heavy and light chain is replaced with a polypeptide having CTLA4 activity creating a CTLA4-Ab molecule. In a preferred embodiment the heavy chain constant region of the CTLA4-Ab molecule comprises a hinge region, a CH2 domain and a CH3 domain derived from Cxcex31, Cxcex32, Cxcex33 or Cxcex34. In a preferred embodiment the light chain constant region of the CTLA4-Ab molecule comprises an Ig signal sequence, the CTLA4 extracellular domain, and the light chain (kappa or lambda) constant domain.
The nucleic acids obtained in accordance with this invention can be inserted into various expression vectors, which in turn direct the synthesis of the corresponding protein in a variety of hosts, particularly eucaryotic cells, such as mammalian or insect cell culture and procaryotic cells, such as E. coli. Expression vectors within the scope of the invention comprise a nucleic acid as described herein and a promotor operably linked to the nucleic acid. Such expression vectors can be used to transfect host cells to thereby produce fusion proteins encoded by nucleic acids as described herein.
Another aspect of the invention pertains to isolated CTLA4-immunoglobulin fusion proteins comprising a first peptide having a CTLA4 activity and a second peptide comprising an immunoglobulin constant region which is modified to reduce at least one constant region-mediated biological effector function relative to a CTLA4-IgG1 fusion protein. A preferred CTLA4-immunoglobulin fusion protein comprises an extracellular domain of the CTLA4 protein (e.g., amino acid positions 20-144 of the human CTLA4-immunoglobulin fusion protein shown in SEQ ID NO: 24, 26 and 28) linked to an immunoglobulin constant region comprising a hinge region, a CH2 domain and a CH3 domain derived from Cxcex31, Cxcex32, Cxcex33 or Cxcex34. A preferred constant domain used to reduce the complement activating ability of the fusion protein is Cxcex34. In one embodiment, the CH2 domain of the immunoglobulin constant region is modified to reduce at least one biological effector function, such as complement activation or Fc receptor interaction. In a particularly preferred embodiment, the CTLA4-immunoglobulin fusion protein includes a CH2 domain which is modified by substitution of an amino acid residue at position 234, 235 and/or 237 of an intact heavy chain protein. One example of such a protein is a CTLA4-immunoglobulin fusion protein fused to IgG4 comprising an amino acid sequence shown in SEQ ID NO: 28 or a CTLA4-immunoglobulin fused to IgG1 fusion protein comprising an amino acid sequence shown in SEQ ID NO: 24.
The CTLA4-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a CTLA4 ligand (e.g., B7-1 and/or B7-2) and a receptor therefor (e.g., CD28 and/or CTLA4) on the surface of a T cell, to thereby suppress cell-mediated immune responses in vivo. Inhibition of the CTLA4 ligand/receptor interaction may be useful for both general immunosuppression and to induce antigen-specific T cell tolerance in a subject for use in preventing transplantation rejection (solid organ, skin and bone marrow) or graft versus host disease, particularly in allogeneic bone marrow transplantation. The CTLA4-immunoglobulin fusion proteins can also be used therapeutically in the treatment of autoimmune diseases, allergy and allergic reactions, transplantation rejection and established graft versus host disease in a subject. Moreover, the CTLA4-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-CTLA4 antibodies in a subject, to purify CTLA4 ligands and in screening assays to identify molecules which inhibit the interaction of CTLA4 with a CTLA4 ligand.